In vitro enzymatic electrochemical monitoring of glucose metabolism and production in rat primary hepatocytes on highly O2 permeable plates.

In situ continuous glucose monitoring under physiological culture conditions is imperative in understanding the dynamics of cell and tissue behaviors and their physiological responses since glucose plays an important role in principal source of biological energy. We therefore examined physiologically relevant dynamic changes in glucose levels based on glucose metabolism and production during aerobic culture (10% O2) of rat primary hepatocytes stimulated with insulin or glucagon on a highly O2 permeable plate, which can maintain the oxygen concentration close to the periportal zone of the liver. As glucose monitoring devices, we used oxygen-independent glucose dehydrogenase-modified single-walled carbon nanotube electrodes placed close to the surface of the hepatocytes. The current response of glucose oxidation slightly decreased after the addition of insulin in the presence of glucose due to the acceleration of glucose uptake by the hepatocytes, whereas that significantly increased after the addition of glucagon and fructose even in the absence of glucose due to the conversion of fructose to glucose based on gluconeogenesis. These phenomena might be consistent relatively with the physiological behaviors of hepatocytes in the periportal region. The present monitoring system would be useful for the studies of glucose homeostasis and diabetes in vitro.

Coculture with hiPS-derived intestinal cells enhanced human hepatocyte functions in a pneumatic-pressure-driven two-organ microphysiological system.

Examining intestine-liver interactions is important for achieving the desired physiological drug absorption and metabolism response in in vitro drug tests. Multi-organ microphysiological systems (MPSs) constitute promising tools for evaluating inter-organ interactions in vitro. For coculture on MPSs, normal cells are challenging to use because they require complex maintenance and careful handling. Herein, we demonstrated the potential of coculturing normal cells on MPSs in the evaluation of intestine-liver interactions. To this end, we cocultured human-induced pluripotent stem cell-derived intestinal cells and fresh human hepatocytes which were isolated from PXB mice with medium circulation in a pneumatic-pressure-driven MPS with pipette-friendly liquid-handling options. The cytochrome activity, albumin production, and liver-specific gene expressions in human hepatocytes freshly isolated from a PXB mouse were significantly upregulated via coculture with hiPS-intestinal cells. Our normal cell coculture shows the effects of the interactions between the intestine and liver that may occur in vivo. This study is the first to demonstrate the coculturing of hiPS-intestinal cells and fresh human hepatocytes on an MPS for examining pure inter-organ interactions. Normal-cell coculture using the multi-organ MPS could be pursued to explore unknown physiological mechanisms of inter-organ interactions in vitro and investigate the physiological response of new drugs.

Bioelectrochemical detection of histamine release from basophilic leukemia cell line based on histamine dehydrogenase-modified cup-stacked carbon nanofibers.

Histamine released from mast cells plays an important role as not only a physiological active substance for the trigger of allergic reactions but also a neurotransmitter in a nervous system. To detect histamine directly, we immobilized oxygen-independent histamine dehydrogenase (HmDH) onto the surface of cup-stacked carbon nanofibers (CSCNFs), which work both as an electrical nanowire and an enzyme support, and investigated the direct electron transfer reaction from HmDH reduced by histamine to CSCNF. Current responses of histamine oxidation at the HmDH-modified CSCNF electrode showed a linear relationship between 0.3 µM and 300 µM with detection limit of 0.1 µM in a Briton Robinson buffer (pH 9.4) and was about 25 times larger than those at a flat glassy carbon electrode modified with HmDH because of its three-dimensional network. Using the HmDH-modified CSCNF electrode, we successfully observed histamine release from rat basophilic leukemia cell line RBL-2H3 after stimulation with degranulation agents, such as antigen 2,4-dinitrophenylated bovine serum albumin and calcium ionophore A23187. The present sensing system might be applied to at least advanced in vitro allergic diagnostic methods.

Direct Electron Transfer Kinetics of Peroxidase at Edge Plane Sites of Cup-Stacked Carbon Nanofibers and Their Comparison with Single-Walled Carbon Nanotubes.

Electron transfer kinetics at the graphene edge site is of great interest from the viewpoints of application to sensing and energy conversion and storage. Here we analyzed kinetics of direct electron transfer of horseradish peroxidase (HRP) adsorbed through surfactant sodium dodecyl sulfate at cup-stacked carbon nanofibers (CSCNFs), which provide highly ordered graphene edges, and compared it with that at single-walled carbon nanotubes (SWCNTs), which consist of a rolled-up basal plane graphene. The heterogeneous electron transfer rate constant of the Fe(2+/3+) couple of the HRP reaction center at CSCNFs (ca. 34.8 s(-1)) was an order of magnitude larger than that at SWCNTs (ca. 4.7 s(-1)). In addition, the overall rate constant of the electron transfer reaction from CSCNFs to HRP oxidized by H2O2 was higher than that from SWCNTs by a factor of 3. CSCNFs also allowed enhancement of the complex-formation reaction rate of HRP with H2O2, in comparison with that at SWCNTs. CSCNFs would therefore be applied to not only biosensors but also biofuel cells with enhanced performance.

Electrochemical properties of seamless three-dimensional carbon nanotubes-grown graphene modified with horseradish peroxidase.

Horseradish peroxidase (HRP) was immobilized through sodium dodecyl sulfate (SDS) on the surface of a seamless three-dimensional hybrid of carbon nanotubes grown at the graphene surface (HRP-SDS/CNTs/G) and its electrochemical properties were investigated. Compared with graphene alone electrode modified with HRP via SDS (HRP-SDS/G electrode), the surface coverage of electroactive HRP at the CNTs/G electrode surface was approximately 2-fold greater because of CNTs grown at the graphene surface. Based on the increase in the surface coverage of electroactive HRP, the sensitivity to H2O2 at the HRP-SDS/CNTs/G electrode was higher than that at the HRP-SDS/G electrode. The kinetics of the direct electron transfer from the CNTs/G electrode to compound I and II of modified HRP was also analyzed.

Electrochemically Functionalized Seamless Three-Dimensional Graphene-Carbon Nanotube Hybrid for Direct Electron Transfer of Glucose Oxidase and Bioelectrocatalysis.

Three-dimensional seamless chemical vapor deposition (CVD) grown graphene-carbon nanotubes (G-CNT) hybrid film has been studied for its potential in achieving direct electron transfer (DET) of glucose oxidase (GOx) and its bioelectrocatalytic activity in glucose detection. A two-step CVD method was employed for the synthesis of seamless G-CNT hybrid film where CNTs are grown on already grown graphene film on copper foil using iron as a catalyst. Physical characterization using SEM and TEM show uniform dense coverage of multiwall carbon nanotubes (MWCNT) grown directly on graphene with seamless contacts. The G-CNT hybrid film was electrochemically modified to introduce oxygenated functional groups for DET favorable immobilization of GOx. Pristine and electrochemically functionalized G-CNT film was characterized by electrochemical impedance spectroscopy (EIS), cyclic voltammetry, X-ray photoelectron-spectroscopy, and Raman spectroscopy. The DET between GOx and electrochemically oxidized G-CNT electrode was studied using cyclic voltammetry which showed a pair of well-defined and quasi-reversible redox peaks with a formal potential of -459 mV at pH 7 corresponding to the redox site of GOx. The constructed electrode detected glucose concentration over the clinically relevant range of 2-8 mM with the highest sensitivity of 19.31 µA/mM/cm(2) compared to reported composite hybrid electrodes of graphene oxide and CNTs. Electrochemically functionalized CVD grown seamless G-CNT structure used in this work has potential to be used for development of artificial mediatorless redox enzyme based biosensors and biofuel cells.

New physiologically-relevant liver tissue model based on hierarchically cocultured primary rat hepatocytes with liver endothelial cells.

To develop an in vitro liver tissue equivalent, hepatocytes should be cocultured with liver non-parenchymal cells to mimic the in vivo physiological microenvironments. In this work, we describe a physiologically-relevant liver tissue model by hierarchically organizing layers of primary rat hepatocytes and human liver sinusoidal endothelial cells (TMNK-1) on an oxygen-permeable polydimethylsiloxane (PDMS) membrane, which facilitates direct oxygenation by diffusion through the membrane. This in vivo-mimicking hierarchical coculture was obtained by simply proceeding the overlay of TMNK-1 cells on the hepatocyte layer re-formed on the collagen immobilized PDMS membranes. The comparison of hepatic functionalities was achieved between coculture and sandwich culture with Matrigel, in the presence and absence of direct oxygenation. A complete double-layered structure of functional liver cells with vertical contact between hepatocytes and TMNK-1 was successfully constructed in the coculture with direct oxygen supply and was well-maintained for 14 days. The hepatocytes in this hierarchical culture exhibited improved survival, functional bile canaliculi formation, cellular level polarization and maintenance of metabolic activities including Cyp1A1/2 activity and albumin production. By contrast, the two cell populations formed discontinuous monolayers on the same surfaces in the non-oxygen-permeable cultures. These results demonstrate that (i) the direct oxygenation through the PDMS membranes enables very simple formation of a hierarchical structure consisting of a hepatocyte layer and a layer of TMNK-1 and (ii) we may include other non-parenchymal cells in this format easily, which can be widely applicable to other epithelial organs.

Bioelectrochemistry of heme peptide at seamless three-dimensional carbon nanotubes/graphene hybrid films for highly sensitive electrochemical biosensing.

A seamless three-dimensional hybrid film consisting of carbon nanotubes grown at the graphene surface (CNTs/G) is a promising material for the application to highly sensitive enzyme-based electrochemical biosensors. The CNTs/G film was used as a conductive nanoscaffold for enzymes. The heme peptide (HP) was immobilized on the surface of the CNTs/G film for amperometric sensing of H2O2. Compared with flat graphene electrodes modified with HP, the catalytic current for H2O2 reduction at the HP-modified CNTs/G electrode increased due to the increase in the surface coverage of HP. In addition, microvoids in the CNTs/G film contributed to diffusion of H2O2 to modified HP, resulting in the enhancement of the catalytic cathodic currents. The kinetics of the direct electron transfer from the CNTs/G electrode to compound I and II of modified HP was also analyzed.

The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas-permeable membranes.

Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%-O2 (+)] or physiological oxygen concentrations [10%-O2 (+), 5%-O2 (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas-impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS-O2 (-)]. The results indicated that the hepatocytes under 10%-O2 (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS-O2 (-) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug-metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long-term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen-permeable membrane system to provide a simple method for in vitro studies.

Electrochemical properties of oxygenated cup-stacked carbon nanofiber-modified electrodes.

Oxygenated cup-stacked carbon nanofibers (CSCNFs), the surface of which provides highly ordered graphene edges and oxygen-containing functional groups, were investigated as electrode materials by using typical redox species in electrochemistry, Fe(2+/3+), [Fe(CN)6](3-/4-), and dopamine. The electron transfer rates for these redox species at oxygenated CSCNF electrodes were higher than those at edge-oriented pyrolytic graphite and glassy carbon electrodes. In addition, the oxygen-containing functional groups also contributed to the electron transfer kinetics at the oxygenated CSCNF surface. The electron transfer rate of Fe(2+/3+) was accelerated and that of [Fe(CN)6](3-/4-) was decelerated by the oxygen-containing groups, mainly due to the electrostatic attraction and repulsion, respectively. The electrochemical reaction selectivities at the oxygenated CSCNF surface were tunable by controlling the amount of nanofibers and the oxygen/carbon atomic ratio at the nanofiber surface. Thus, the oxygenated CSCNFs would be useful electrode materials for energy-conversion, biosensing, and other electrochemical devices.

Combination of microwell structures and direct oxygenation enables efficient and size-regulated aggregate formation of an insulin-secreting pancreatic ß-cell line.

Spherical three-dimensional (3D) cellular aggregates are valuable for various applications such as regenerative medicine or cell-based assays due to their stable and high functionality. However, previous methods to form aggregates have shown drawbacks, being labor-intensive, showing low productivity per unit area or volume and difficulty to form homogeneous aggregates. We proposed a novel strategy based on oxygen-permeable polydimethylsiloxane (PDMS) honeycomb microwell sheets, which can theoretically supply about 80 times as much oxygen as conventional polystyrene culture dishes, to produce recoverable aggregates in controllable sizes using mouse insulinoma cells (MIN6-m9). In 48 hours of culture, the PDMS sheets produced aggregates whose diameters were strictly controlled (≃32, 60, 90, 150 and 280 mm) even at an inoculum density eight times higher (8.0×105 cells/cm(2) ) than that of normal confluent monolayers (1.0×105 cells/cm(2) ). Measurement of the oxygen tension near the cell layer and glucose/lactate analysis clearly showed that cells exhibit aerobic respiration on the PDMS-based culture system. Glucose-responsive insulin secretion of the recovered aggregates showed that the aggregates around 90 mm in diameter secreted the largest amounts of insulin. This confirmed the advantages of 3D cellular organization and the existence of a suitable aggregate size, above which excess organization leads to a decreased metabolic response. These results demonstrated that this microwell-based PDMS culture system provides a promising method to form size-regulated and better functioning 3D cellular aggregates of various kinds of cells with a high yield per surface area.

Formation and harvesting of thick pancreatic ß-cell sheets on a highly O2-permeable plate modified with poly(N-isopropylacrylamide).

Producing sheet-like tissues is a promising strategy for implantable engineered tissues, because in vitro pre-vascularization is dispensable in this configuration. We developed a simple methodology for the formation and non-destructive harvesting of a thick pancreatic ß-cell sheet consisting of mouse insulinoma MIN6-m9 cells and mouse NIH3T3 fibroblasts using an O2-permeable polydimethylsiloxane plate modified with poly(N-isopropylacrylamide) (O2+/PNIPA-PDMS plate). Owing to the direct oxygenation of the cells through the PNIPA-modified PDMS plate, a viable, metabolically active sheet 5-6 cell layers thick (ca. 60 µm thick) was formed spontaneously; in the absence of direct oxygenation, only a thin cell sheet could be formed consisting of at most 2 layers (ca. 20 µm thick) with mainly anaerobic metabolism. Consequently, the net density of MIN6-m9 cells under direct oxygenation was about twice as high as in the absence of direct oxygenation. Accordingly, the insulin secretion for 10 to 60 min after glucose stimulation was also about 1.5 times higher with oxygenation. Furthermore, the thick cell sheet was successfully harvested from the O2+/PNIPA-PDMS plate surface in a non-destructive manner by inducing a phase transition of PNIPA by lowering the temperature below the lower critical solution temperature. Thus, the present report shows a promising and simple method to produce thick sheet-like engineered tissues for transplantation that could be used as a treatment for type 1 diabetes.

Direct synthesis of cup-stacked carbon nanofiber microspheres by the catalytic pyrolysis of poly(ethylene glycol).

Uniformly sized microspheres tangled with cup-stacked carbon nanofibers (CSCNFs) were directly synthesized by the pyrolysis of poly(ethylene glycol) (PEG) with a nickel catalyst. A PEG/Ni membrane was prepared on a silicon wafer surface by heating it to 750 °C at a heating rate of 15 °C min(-1). The wafer was heated to a temperature of 400 °C and was held at that temperature for 1 h before raising the temperature to 750 °C for 10 min to form the CSCNF microspheres. The final CSCNF microspheres and the intermediates were evaluated using scanning electron microscopy, transmission electron microscopy, X-ray diffractometry, and Raman spectroscopy to elucidate the growth mechanism. Furthermore, the CSCNF microspheres were successfully dispersed and maintained their spherical shape in an aqueous solution containing 0.5% Nafion. The CSCNF microspheres have the potential to work as a sophisticated carrier with high adsorption and fast electron-transfer exchange properties based on the graphene edges of the nanofiber surface.

Phthalocyanine-based fluorescence probes for detecting ascorbic acid: phthalocyaninatosilicon covalently linked to TEMPO radicals.

We have applied phthalocyaninatosilicon (SiPc) covalently linked to one or two tetramethyl-1-piperidinyloxyl (TEMPO) radicals as fluorescence probes for detecting ascorbic acid in biological systems.

Cytotoxicity evaluation of reactive metabolites using rat liver homogenate microsome-encapsulated alginate gel microbeads.

We present an improved cytotoxicity test for reactive metabolites, in which the S9 microsomal fraction of rat liver homogenate is encapsulated in alginate gel microbeads to avoid cytotoxic effects of S9-self-generated toxicants, microsomal lipid peroxides. The S9-encapsulated gel microbeads were prepared by a coaxial two-fluid nozzle and surfaces of the microbeads were coated with poly-L-lysine (PLL). Although the initial metabolic rate of the S9-encapsulated gel microbeads was about 20% slower than that of bare S9, the microbeads prevented the leakage of microsomal lipid peroxides thanks to the dense alginate and PLL polymer networks. In fact, the half maximal effective concentration of the indirect mutagen cyclophosphamide on NIH3T3 cells in the presence of the S9-encapsulated gel microbeads was about 5 times higher than that in the presence of bare S9. Use of the S9-encapsulated gel microbeads enabled the more accurate evaluation of the cytotoxicity of the reactive metabolites without the S9-based cytotoxicity.

Electrochemical biosensor for the detection of H2O2 from living cancer cells based on ZnO nanosheets.

In this work, direct electron transfer of cytochrome c (cyt. c)--a model for studying the electron transfer of enzymes is achieved at hexagonal ZnO nanosheets by one-step electrodeposition. UV-vis spectra and electrochemical data demonstrate that such ZnO nanosheets can supply a bio-compatible surface to keep the bioactivity of cyt. c. The redox formal potential (E(0)') of cyt. c is estimated to be 338.2+/-4.3 mV (vs. AgAgCl) at the nanostructured ZnO surface. This value is much more positive than those of enzymes previously obtained at other metal oxides and zeolite surfaces. Experiment data show, under the optimized potential of 0.0 V (vs. AgAgCl), the electrochemical determination of H(2)O(2) is free from not only anodic interferences like ascorbic acid (AA) and dopamine (DA), but also a cathodic interference-O(2). Such an excellent selectivity enable the present H(2)O(2) biosensor determine the extracellular H(2)O(2) released from living human hepatoma cells.

A micropatterned cell array with an integrated oxygen-sensitive fluorescent membrane.

We propose a simple method for producing micropatterned cell spots by photocatalytic lithography on a Pt porphyrin-based oxygen-sensitive polystyrene membrane that enables real-time imaging of oxygen consumption of patterned cell spots with sub-millimetre resolution.

Simultaneous evaluation of toxicities using a mammalian cell array chip prepared by photocatalytic lithography.

A prototype of a mammalian cell array chip was developed on a flat glass surface. A superhydrophilic (water contact angle=5 degrees)/highly hydrophobic (120 degrees) pattern was prepared on a fluorinated polymer-coated glass surface by means of photocatalytic lithography, and A549 (a human alveolar epithelial cell line), Hep G2 (a human hepatoma cell line) and mouse fibroblast 3T3 cells were inoculated onto the superhydrophilic regions. The cell populations were confined in the superhydrophilic regions for at least 24 h and separated from each other for at least one week. Organ-specific toxicity of aflatoxin B(1) and non-specific toxicity of adriamycin were successfully detected by using the cell array chip.

A rapid and simple evaluation system for gas toxicity using luminous bacteria entrapped by a polyion complex membrane.

We have developed a rapid and simple gas toxicity evaluation system based on bioluminescence inhibition of a marine-derived wild luminous bacterium, Vibrio fischeri. The luminous bacteria were trapped using a thin polyion complex membrane in order to allow semi direct contact between the bacteria and toxic gases. Bioluminescence inhibition ratios of the present system to six reference gases, including benzene, trichloroethylene, acetone, NO(2), SO(2), and CO, were evaluated, and dose-response relationships were successfully obtained after 15 min of gas exposure, except for CO gas. The sensitivity to the five gases except for CO gas of the present system was 1-3 orders of magnitude higher than that in acute animal tests. The present system also allowed for the evaluation of overall toxicity of some environmental gases containing various chemicals. These results clearly demonstrated that the present system would be a valuable prototype for rapid and on-site acute toxicity detection of a gas mixture, such as environmental gases.

Development of an in vitro batch-type closed gas exposure device with an alveolar epithelial cell line, A549, for toxicity evaluations of gaseous compounds.

To simply evaluate toxicity for various types of exhaust-gas samples collected in various locations, we developed a small-scale (150 mL) batch-type completely closed gas exposure device incorporated with an air-liquid interface culture of a human alveolar epithelial cell line, A549. On the basis of cell viability tests using an acid phosphatase assay after 48 h of gas exposure, the developed device was able to measure clear dose-response relationships for volatile organic and inorganic compounds, such as benzene, trichloroethylene (TCE), acetone, SO(2) and NO(2) gases, but not CO gas. Although the 50% effective concentration values in the device were much higher than 50% lethal concentration values reported in animal experiments, the tendency of the toxic intensity observed in the former was roughly consistent with that of the acute toxicity in the latter. We further applied the device to evaluate the toxicity of cigarette smoke as an example of actual environmental gases, and successfully measured acute cell death from the gas after 48 h of exposure. The present small device is expected to be one of good tools not only in simultaneously assessing various gaseous chemicals or samples, but also in studying acute toxicity expression mechanisms in human lung epithelia.